Quinazolinone derivatives for use in the treatment of chlamydiales infections

ABSTRACT

The present invention relates to a method for treating a Chlamydiae infection comprising the administration of a therapeutically effective amount of a compound of formula (I) to a subject in need thereof: 
                         
Wherein, R1, R2, R3, R4 and p are as defined in claim  1.

The present invention is in the field of therapeutic drugs to treatintracellular bacterial infection and disease. In particular, theinvention provides quinazolinone compounds for use in the treatment ofinfections by pathogenic intracellular bacteria in the orderChlamydiales.

The bacterial order Chlamydiales includes only obligately intracellularbacteria that have a chlamydia-like developmental cycle of replication.Chlamydiales live in animals, insects, and protozoa. The orderChlamydiales belongs to the class Chlamydiae, phylum Chlamydiae, domainBacteria.

Chlamydiales order display a stereotypical developmental cycle thatalternates between two forms. The elementary body (EB) is the infectiousform that attaches to and invades target epithelial cells. After entry,the EB form transitions to a reticulate body (RB), which proliferateswithin the expanding parasitophorous vacuole, termed the inclusion(Field & Hackstadt 2002). Upon triggering by an undefined signal, RBstransition to infectious progeny, which are later released to thesurrounding milieu either by lysis or an extrusion mechanism, to infectnew host cells (Hybiske & Stephens 2007).

Currently, the order Chlamydiales includes the families Chlamydiaceae,Simkaniaceae and Waddliaceae, which have Gram-negative extracellularinfectious bodies (EBs), and Parachlamydiaceae, which has variable Gramstaining of EBs.

Mammalian pathogenic bacteria from Chlamydiales order include Chlamydiatrachomatis responsible for genital, ocular and lung infections;Chlamydophila pneumoniae responsible for a pneumonia and associated withasthma; Chlamydophila psittaci responsible for a pneumonia transmittedto humans by birds; Simkania negevensis is associated with infections ofthe upper respiratory tract in infants and adults.

Chlamydia trachomatis (Ctr) is an obligate intracellular human pathogenand one of the main causative agents of sexually transmitted diseases(STD). Infections of the eye with Ctr can lead to chronic conjunctivitis(trachoma) resulting in preventable blindness if untreated. The WHOestimates around 540 million people suffering from Ctr ocular infectionwhereby 1.2 million people developed blindness (Resnikoff, S., et al.,Global data on visual impairment in the year 2002. Bull World HealthOrgan, 2004. 82(11): p. 844-51).

Alternatively, infections of the urogenital tract cause prostatitis,pelvic inflammatory disease and in women increased risk of ectopicpregnancy or infertility. The number of urogenital tract infections withCtr worldwide in 2008 was estimated by the WHO to about 106 millioncases.

As many as half of all infants born to mothers with chlamydia will beborn with the disease. Chlamydia can affect infants by causingspontaneous abortion; premature birth; conjunctivitis, which may lead toblindness; and pneumonia. Chlamydia may also cause reactive arthritis(Reiters' syndrome)—the triad of arthritis, conjunctivitis andurethritis (inflammation of the urethra)—especially in young men. About15,000 men develop reactive arthritis due to chlamydia infection eachyear in the U.S., and about 5,000 are permanently affected by it. It canoccur in both sexes, though is more common in men.

Although antibiotics resistance is considered rare for Chlamydiatrachomatis, it is widespread in several sexually transmitted bacteria,e.g. Neisseria gonorrhoeae or Mycoplasma genitalium (Unemo, M. and W. M.Shafer, Antibiotic resistance in Neisseria gonorrhoeae: origin,evolution, and lessons learned for the future. Ann NY Acad Sci, 2011.1230: p. E19-28). The rapid spread of Azithromycin resistances amongthese bacteria has been attributed to the frequent treatment of patientswith chlamydial infection with this antibiotic (Unemo, M. and W. M.Shafer, Antibiotic resistance in Neisseria gonorrhoeae: origin,evolution, and lessons learned for the future. Ann NY Acad Sci, 2011.1230: p. E19-28; Ison, C., Antimicrobial resistance in sexuallytransmitted infections in the developed world: implications for rationaltreatment. Curr Opin Infect Dis., 2012). Development of treatmentregimens more specific for particular pathogens without affecting othershas been suggested as one strategy to avoid the continuous spread ofantibiotic resistances.

Species in the family Simkaniaceae Parachlamydiaceae and Waddliaceaehave a chlamydia-like cycle of replication. They may infect humans andgive respiratory diseases; they currently include two genera: Simkaniaand Fritschea.

Parachlamydiaceae such as Parachlamydia acanthamoebae have been found inthe respiratory tract of humans and could be important respiratorypathogens. Waddliaceae can provoke abortion in ruminants. Two Fritscheaspecies have been identified in insects. Piscichlamydia salmonis hasrecently been identified as an agent of the gill epitheliocystis in theAtlantic salmon.

The genome of Simkania negevensis (Sn) is approximately 2.5 Mbp in sizeand thus 2-3 times larger than the genome of Chlamydia (Collingro A,Tischler P, Weinmaier T, Penz T, Heinz E, Brunham R C, Read T D, BavoilP M, Sachse K, Kahane S, Friedman M G, Rattei T, Myers G S, Horn M.Unity in variety—the pan-genome of the Chlamydiae. Mol Biol Evol 2011;28(12):3253-3270). Sn is able to replicate in several amoebae, human andsimian epithelial cells and macrophages (Kahane S, Fruchter D, DvoskinB, Friedman M G. Versatility of Simkania negevensis infection in vitroand induction of host cell inflammatory cytokine response. J Infect2007; 55(2):e13-21; Kahane S, Gonen R, Sayada C, Elion J, Friedman M G.Description and partial characterization of a new Chlamydia-likemicroorganism. FEMS Microbiol Lett 1993; 109(2-3):329-333) and has beenassociated with infections of the upper respiratory tract in infants andadults (Horn M. Chlamydiae as Symbionts in Eukaryotes. Annu RevMicrobiol 2008; 62:113-131; Kahane S, Greenberg D, Friedman M G, HaikinH, Dagan R. High prevalence of “Simkania Z” a novel Chlamydia-likebacterium in infants with acute bronchiolitis. (vol 177, pg 1425, 1998).J Infect Dis 1998; 178(5):1553-1553; Lieberman D, Kahane S, Lieberman D,Friedman M G. Pneumonia with serological evidence of acute infectionwith the Chlamydia-like microorganism “Z”. Am J Respir Crit Care Med1997; 156(2 Pt 1):578-582; Lamoth F, Greub G. Amoebal pathogens asemerging causal agents of pneumonia. Fems Microbiol Rev 2010;34(3):260-280).

Infections with the two closely related human pathogenic bacteriaChlamydophila pneumoniae and Chlamydophila psittaci can cause communityacquired or animal transmitted pneumonia, chronic bronchitis and chronicasthma (Harkinezhad T, Geens T, Vanrompay D. Chlamydophila psittaciinfections in birds: A review with emphasis on zoonotic consequences.Vet Microbiol 2009; 135(1-2):68-77; Hughes C, Maharg P, Rosario P,Herrell M, Bratt D, Salgado J, Howard D. Possible nosocomialtransmission of psittacosis. Infect Control Hosp Epidemiol 1997;18(3):165-168; Hahn D L, McDonald R. Can acute Chlamydophila pneumoniaerespiratory tract infection initiate chronic asthma, Ann Allergy AsthmaImmunol 1998; 81(4):339-344).

Accordingly, it is an object of the present invention to providecompounds alternative to conventional antibiotics useful for preventingand/or treating infections by bird, fish and mammalian, preferably humanand zoonotic, pathogenic Chlamydiales.

Inventors have for the first time demonstrated that a selection ofcycloamine derivatives shows a strong inhibition of infections withpathogenic bacteria in the order of Chlamydiales.

In particular, Inventors have found that compounds of formula (I′) aresurprisingly highly potent at low concentration and affect cellularmechanisms that are important at defined stages of the chlamydialdevelopmental cycle.

Compounds of Formula (I) for Use in the Treatment of ChlamidyalesInfection

Thus, in one aspect, the present invention relates to a compound offormula (I)

-   -   Wherein:    -   R₁ is at each occurrence, independently selected from H, Cl, Br,        I, F;    -   R₂ is independently selected from H, Cl, Br, F, I, C₁-C₆ alkyl,        C₆-C₁₀ aryl, 5 to 10 membered heteroaryl, said aryl or        heteroaryl groups being optionally substituted by one to three        R₅;    -   R₃ is independently selected from H, F, Cl, Br, I, C₁-C₆ alkyl;    -   R₄ is, at each occurrence, independently selected from H, C₁-C₆        alkyl;    -   R₅ is independently selected from F, Cl, Br, I, C₁-C₆ alkyl,        C₆-C₁₀ aryl, said aryl group being optionally substituted by one        to three R₆;    -   R₆ is F, Cl, Br, I;    -   p is 1, 2 or 3,    -   and the stereoisomeric forms, mixtures of stereoisomeric forms        or pharmaceutically acceptable salts forms thereof,    -   for use in the treatment of a Chlamydiae infection.

In another aspect, the present invention relates to a compound offormula (I)

-   -   Wherein:    -   R₁ is at each occurrence, independently selected from H, Cl, Br,        I, F;    -   R₂ is independently selected from H, Cl, Br, F, I, C₁-C₆ alkyl,        C₆-C₁₀ aryl, 5 to 10 membered heteroaryl, said aryl or        heteroaryl groups being optionally substituted by one to three        R₅;    -   R₃ is independently selected from H, F, Cl, Br, I, C₁-C₆ alkyl;    -   R₄ is, at each occurrence, independently selected from H, C₁-C₆        alkyl;    -   R₅ is independently selected from F, Cl, Br, I, C₁-C₆ alkyl,        C₆-C₁₀ aryl, said aryl group being optionally substituted by one        to three R₆;    -   R₆ is F, Cl, Br, I;    -   p is 1, 2 or 3,    -   and the stereoisomeric forms, mixtures of stereoisomeric forms        or pharmaceutically acceptable salts forms thereof,    -   providing said compound is not of the following formula:

-   -   for use in the treatment of a Chlamydiae infection.

In a preferred embodiment, the Chlamydiae infection is a Chlamydia orSimkania infection.

In another embodiment, there are included compounds of formula (I) foruse as defined above, wherein at least one of R₁ is H or F.

In still another embodiment, there are included compounds of formula(I′) for use as defined above, which are compounds of formula (I)wherein R₂ is independently selected from H, Cl, Br, F, I, C₆-C₁₀ aryl,5 to 10 membered heteroaryl, said aryl or heteroaryl groups beingoptionally substituted by one to three R₅, R₅ being as defined above.

In still another embodiment, there are included compounds of formula (I)for use as defined above, wherein R₂ is a 5 to 10 membered heteroaryl.

In yet another embodiment, there are included compounds of formula (I)for use as defined above, wherein R₂ is 1,3-thiazolyl, preferably4-(1,3-thiazolyl).

In an additional embodiment, there are included compounds of formula (I)for use as defined above, wherein R₃ is H.

In an additional embodiment, there are included compounds of formula (I)for use as defined above, wherein R₄ is H.

In another additional embodiment, there are included compounds offormula (I) for use as defined above, wherein R₄ is C₁-C₆ alkyl, notablymethyl.

In a particular embodiment, there are included compounds of formula (I)for use as defined above, which are selected from:

Retro-2 2-(5-methylthiophen-2-yl)-3- phenyl-2,3-dihydroquinazolin-4(1H)-one

Retro-2.1 6-fluoro-1-methyl-2-(5-(2- methylthiazol-4-yl)thiophen-2-yl)-3-phenyl-2,3- dihydroquinazolin-4(1H)-one

compound 25 2-(5-(2-methylthiazol-4- yl)thiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one

RN-2-016 2-(5-bromothiophen-2-yl)-1- methyl-3-phenyl-2,3-dihydroquinazolin-4(1H)-one

RN-3-072 3-(2-chlorophenyl)-1-methyl-2- (5-(2-methylthiazol-4-yl)thiophen-2-yl)-2,3- dihydroquinazolin-4(1H)-one

VP89 2-(5-(2-(4-fluorophenyl)thiazol- 4-yl)thiophen-2-yl)-1-methyl-3-phenyl-2,3-dihydroquinzolin- 4(1H)-one

In a particular embodiment, there are included compounds of formula (I)for use as defined above, which are selected from:

-   6-fluoro-1-methyl-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one    (Retro 2.1)-   2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one    (Compound 25)-   2-(5-bromothiophen-2-yl)-1-methyl-3-phenyl-2,3-dihydroquinazolin-4(1H)-one    (RN 2-016)-   3-(2-chlorophenyl)-1-methyl-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-2,3-dihydroquinazolin-4(1H)-one    (RN 3-072)-   2-(5-(2-(4-fluorophenyl)thiazol-4-yl)thiophen-2-yl)-1-methyl-3-phenyl-2,3-dihydroquinazolin-4(1H)-one    (VP 89).

The present invention also relates to a method of treatment comprisingthe administration of a therapeutically effective amount of a compoundof formula (I) to a subject in need thereof.

The compounds of formula (I) are useful for the treatment of infectionwith pathogenic bacteria, preferably mammalian pathogenic bacteria, morepreferably human pathogenic bacteria, in the order of Chlamydiales; inparticular, for the treatment of infection with bacteria in the genus ofChlamydia, such as Chlamydia trachomatis, Chlamydophila pneumoniae,Chlamydophila psyttaci . . . , or Symkania, such as Symkania negevensis. . . .

According to one embodiment, the present invention relates to compoundsof formula (I) for their use for the treatment of:

-   -   respiratory tract infection, such as bronchiolitis, pneumonia,        bronchitis, asthma, with Chlamydophila pneumoniae, Chlamydophila        psyttaci (responsible for a pneumonia transmitted to humans by        birds) and/or Symkania negevensis (infections of the upper        respiratory tract in infants and adults); lung infection with        Chlamydia trachomatis;    -   ocular infection, such as conjunctivitis, with Chlamydia        trachomatis;    -   urogenital tract infection, such as prostatitis, pelvic        inflammation, urethritis, sexually transmitted disease (STD)        with Chlamydia trachomatis;    -   arthritis;        and for the prevention of:    -   blindness induced by ocular infection with Chlamydia        trachomatis;    -   ectopic pregnancy, infertility, spontaneous abortion, premature        birth induced by urogenital tract infection with Chlamydia        trachomatis.

According to another embodiment, the present invention relates tocompounds of formula (I) for their use to inhibit primary and progenyinfection of Symkania.

A primary infection is the initial infection of a host by a pathogen,here infectious bacteria in the order of Chlamydiales.

A progeny infection is the infection of new host cells by the infectiousbacteria released after a previous primary infection of a host cell.

According to a further embodiment, the present invention relates tocompounds of formula (I) for their use to inhibit primary and progenyinfection of Chlamydia.

Pharmaceutical Compositions

In a second aspect, the present invention relates to a pharmaceuticalcomposition comprising a compound of formula (Ia):

-   -   Wherein:    -   R₁, R₃, R₄, R₆, and p are as defined in formula (I) above,    -   R₄ is independently selected from H or C₁-C₆ alkyl,    -   and the stereoisomeric forms, mixtures of stereoisomeric forms        or pharmaceutically acceptable salts forms thereof,    -   in admixture with one or more pharmaceutically acceptable        excipients.

In one embodiment, there are included pharmaceutical compositionswherein, in formula (Ia), R₄ is C₁-C₆ alkyl, notably methyl.

In another embodiment, there are included pharmaceutical compositionswherein, in formula (Ia), R₄ is H.

In another embodiment, there are included pharmaceutical compositionswherein, in formula (Ia), wherein R₃ is H.

In still another embodiment, there are included pharmaceuticalcompositions wherein, in formula (Ia), R₁ is H. In yet anotherembodiment, there are included pharmaceutical compositions wherein thecompound of formula (Ia) is:2-(5-(2-(4-fluorophenyl)thiazol-4-yl)thiophen-2-yl)-1-methyl-3-phenyl-2,3-dihydroquinazolin-4(1H)-one(VP89).

The compounds of formula (I) and/or (Ia) of the present invention may beadministered in the form of a conventional pharmaceutical composition byany route including orally, intramuscularly, subcutaneously, topically,intranasally, intraperitoneally, intrathoracially, intravenously,epidurally, intrathecally, intracerebroventricularly and by injectioninto the joints.

The dosage will depend on the route of administration, the severity ofthe disease, age and weight of the patient and other factors normallyconsidered by the attending physician, when determining the individualregimen and dosage level at the most appropriate for a particularpatient.

For preparing pharmaceutical compositions from the compounds of thepresent invention, inert, pharmaceutically acceptable carriers can beeither solid or liquid. Solid form preparations include powders,tablets, dispersible granules, capsules, cachets, and suppositories.

A solid carrier can be one or more substances, which may also act asdiluents, flavouring agents, solubilizers, lubricants, suspendingagents, binders, or tablet disintegrating agents; it can also be anencapsulating material.

Tablets, powders, cachets, and capsules can be used as solid dosageforms suitable for oral administration.

Liquid form compositions include solutions, suspensions, and emulsions.For example, sterile water or propylene glycol solutions of the activecompounds may be liquid preparations suitable for parenteraladministration. Liquid compositions can also be formulated in solutionin aqueous polyethylene glycol solution.

Aqueous solutions for oral administration can be prepared by dissolvingthe active component in water and adding suitable colorants, flavouringagents, stabilizers, and thickening agents as desired. Aqueous solutionsfor oral use can be made by dispersing the finely divided activecomponent in water together with a viscous material such as naturalsynthetic gums, resins, methyl cellulose, sodium carboxymethylcellulose, and other suspending agents known to the pharmaceuticalformulation art.

Depending on the mode of administration, the pharmaceutical compositionwill according to one embodiment of the present invention include 0.05%to 99% weight (percent by weight), according to an alternativeembodiment from 0.10 to 50% weight, of the compound of the presentinvention, all percentages by weight being based on total composition. Atherapeutically effective amount for the practice of the presentinvention may be determined, by the use of known criteria including theage, weight and response of the individual patient, and interpretedwithin the context of the disease which is being treated or which isbeing prevented, by one of ordinary skills in the art.

Compounds of Formula (Ia)

In a third aspect, the present invention relates to a compound offormula (Ia) as defined above, and the stereoisomeric forms, mixtures ofstereoisomeric forms or pharmaceutically acceptable salts forms thereof.

It is to be noted that the present invention includes the combination ofall the embodiments listed hereabove for formulae (I) or (Ia),respectively.

Definitions

The following terms and expressions contained herein are defined asfollows:

As used herein, a range of values in the form “x-y” or “x to y”, or “xthrough y”, include integers x, y, and the integers therebetween. Forexample, the phrases “1-6”, or “1 to 6” or “1 through 6” are intended toinclude the integers 1, 2, 3, 4, 5, and 6. Preferred embodiments includeeach individual integer in the range, as well as any subcombination ofintegers. For example, preferred integers for “1-6” can include 1, 2, 3,4, 5, 6, 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 2-6, etc.

As used herein, the term “alkyl” refers to a straight-chain, or branchedalkyl group having 1 to 8 carbon atoms, such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl,neopentyl, 1-ethylpropyl, 3-methylpentyl, 2,2-dimethylbutyl,2,3-dimethylbutyl, hexyl, octyl, etc. The alkyl moiety ofalkyl-containing groups, such as alkoxy, alkoxycarbonyl, andalkylaminocarbonyl groups, has the same meaning as alkyl defined above.Lower alkyl groups, which are preferred, are alkyl groups as definedabove which contain 1 to 4 carbons. A designation such as “C₁-C₄ alkyl”refers to an alkyl radical containing from 1 to 4 carbon atoms.

As used herein, the term “aryl” refers to a substituted orunsubstituted, mono- or bicyclic hydrocarbon aromatic ring system having6 to 10 ring carbon atoms. Examples include phenyl and naphthyl.Preferred aryl groups include unsubstituted or substituted phenyl andnaphthyl groups. Included within the definition of “aryl” are fused ringsystems, including, for example, ring systems in which an aromatic ringis fused to a cycloalkyl ring. Examples of such fused ring systemsinclude, for example, indane, indene, and tetrahydronaphthalene.

As used herein, the term “heteroaryl” refers to an aromatic groupcontaining 5 to 10 ring carbon atoms in which one or more ring carbonatoms are replaced by at least one hetero atom such as —O—, —N—, or —S—.Examples of heteroaryl groups include pyrrolyl, furanyl, thienyl,pirazolyl, imidazolyl, thiazolyl, isothiazolyl, isoxazolyl, oxazolyl,oxathiolyl, oxadiazolyl, triazolyl, oxatriazolyl, furazanyl, tetrazolyl,pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, indolyl,isoindolyl, indazolyl, benzofuranyl, isobenzofuranyl, purinyl,quinazolinyl, quinolyl, isoquinolyl, benzoimidazolyl, benzothiazolyl,benzothiophenyl, thianaphthenyl, benzoxazolyl, benzisoxazolyl,cinnolinyl, phthalazinyl, naphthyridinyl, and quinoxalinyl. Includedwithin the definition of “heteroaryl” are fused ring systems, including,for example, ring systems in which an aromatic ring is fused to aheterocycloalkyl ring. Examples of such fused ring systems include, forexample, phthalamide, phthalic anhydride, indoline, isoindoline,tetrahydroisoquinoline, chroman, isochroman, chromene, and isochromene.

As used herein, the term “subject” refers to a warm blooded animal suchas a mammal, preferably a human, or a human child, which is afflictedwith, or has the potential to be afflicted with one or more diseases andconditions described herein.

As used herein, a “therapeutically effective amount” refers to an amountof a compound of the present invention effective to prevent or treat thesymptoms of particular disorder.

As used herein, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem complicationscommensurate with a reasonable benefit/risk ratio.

All other terms used in the description of the present invention havetheir meanings as is well known in the art.

In another aspect, the present invention is directed to pharmaceuticallyacceptable salts of the compounds described above. As used herein,“pharmaceutically acceptable salts” includes salts of compounds of thepresent invention derived from the combination of such compounds withnon-toxic acid.

Acid addition salts include inorganic acids such as hydrochloric,hydrobromic, hydroiodic, sulfuric, nitric and phosphoric acid, as wellas organic acids such as acetic, citric, propionic, tartaric, glutamic,salicylic, oxalic, methanesulfonic, para-toluenesulfonic, succinic, andbenzoic acid, and related inorganic and organic acids.

In addition to pharmaceutically-acceptable salts, other salts areincluded in the invention. They may serve as intermediates in thepurification of the compounds, in the preparation of other salts, or inthe identification and characterization of the compounds orintermediates.

The pharmaceutically acceptable salts of compounds of the presentinvention can also exist as various solvates, such as with water,methanol, ethanol, dimethylformamide, ethyl acetate and the like.Mixtures of such solvates can also be prepared. The source of suchsolvate can be from the solvent of crystallization, inherent in thesolvent of preparation or crystallization, or adventitious to suchsolvent. Such solvates are within the scope of the present invention.

It is recognized that compounds of the present invention may exist invarious stereoisomeric forms. As such, the compounds of the presentinvention include both diastereomers and enantiomers. The compounds arenormally prepared as racemates and can conveniently be used as such, butindividual enantiomers can be isolated or synthesized by conventionaltechniques if so desired. Such racemates and individual enantiomers andmixtures thereof form part of the present invention.

It is well known in the art how to prepare and isolate such opticallyactive forms. Specific stereoisomers can be prepared by stereospecificsynthesis using enantiomerically pure or enantiomerically enrichedstarting materials. The specific stereoisomers of either startingmaterials or products can be resolved and recovered by techniques knownin the art, such as resolution of racemic forms, normal, reverse-phase,and chiral chromatography, recrystallization, enzymatic resolution, orfractional recrystallization of addition salts formed by reagents usedfor that purpose. Useful methods of resolving and recovering specificstereoisomers described in Eliel, E. L.; Wilen, S. H. Stereochemistry ofOrganic Compounds; Wiley: New York, 1994, and Jacques, J, et al.Enantiomers, Racemates, and Resolutions; Wiley: New York, 1981, eachincorporated by reference herein in their entireties.

Synthesis

The compounds of the present invention may be prepared in a number ofmethods well known to those skilled in the art, including, but notlimited to those described below, or through modifications of thesemethods by applying standard techniques known to those skilled in theart of organic synthesis. The appropriate modifications andsubstitutions will be readily apparent and well known or readilyobtainable from the scientific literature to those skilled in the art.In particular, such methods can be found in R. C. Larock, ComprehensiveOrganic Transformations, Wiley-VCH Publishers, 1999.

All processes disclosed in association with the present invention arecontemplated to be practiced on any scale, including milligram, gram,multigram, kilogram, multikilogram or commercial industrial scale.

It will be appreciated that the compounds of the present invention maycontain one or more asymmetrically substituted carbon atoms, and may beisolated in optically active or racemic forms. Thus, all chiral,diastereomeric, racemic forms, isomeric forms of a structure areintended, unless the specific stereochemistry or isomeric form isspecifically indicated. It is well-known in the art how to prepare andisolate such optically active forms. For example, mixtures ofstereoisomers may be separated by standard techniques including, but notlimited to, resolution of racemic forms, normal, reverse-phase, andchiral chromatography, preferential salt formation, recrystallization,and the like, or by chiral synthesis either from chiral startingmaterials or by deliberate synthesis of target chiral centers.

Compounds of the present invention may be prepared by a variety ofsynthetic routes. The reagents and starting materials are commerciallyavailable, or readily synthesized by well-known techniques by one ofordinary skill in the arts. All substituents, unless otherwiseindicated, are as previously defined.

In the reactions described hereinafter, it may be necessary to protectreactive functional groups, for example hydroxy, amino, imino, thio orcarboxy groups, where these are desired in the final product, to avoidtheir unwanted participation in the reactions. Conventional protectinggroups may be used in accordance with standard practice, for examplessee T. W. Greene and P. G. M. Wuts in Protective Groups in OrganicChemistry, 3^(rd) ed., John Wiley and Sons, 1999; J. F. W. McOmie inProtective Groups in Organic Chemistry, Plenum Press, 1973.

The general routes to prepare the examples of the present invention areshown in the Scheme A hereafter. The reagents and starting materials arecommercially available, or readily synthesized by well-known techniquesby one of ordinary skill in the arts. All substituents in the syntheticSchemes, unless otherwise indicated, are as previously defined.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of Retro-2 on the Sn bacterial load of infectedHeLa cells by bacterial GroEL immunoblot and Actin used as a loadingcontrol.

FIG. 2 shows the effect of Retro-2 on the Sn inclusion size duringprimary infection of HeLa cells.

FIG. 3 shows the effect of Retro-2 on the number of Sn inclusions duringprogeny infection of HeLa cells

FIG. 4 shows pictures illustrating effect of Retro-2 on phenotypicvariations in Sn inclusion formation in infected HeLa cells.

FIG. 5 shows pictures of the subcellular structure of Sn inclusions ininfected HeLa cells by transmission electron microscopy in presence ofDMSO or of Retro-2.

FIG. 6 shows immunotblot analysis of lysed HeLa cells after Ctr primaryand progeny infection in presence of Retro-2; Chlamydial growth wasdetected with antibodies against chlamydial HSP60 protein and Actin wasused as loading control.

FIG. 7 shows pictures of Ctr infected HeLa cells after Alexa-680 coupledPhalloidin staining; Ctr are detected by GFP-signal.

FIG. 8 shows microscopy images of cells stained for DAPI and Chlamydiatrachomatis (detected by GFP-signal) after Retro-2 and Retro-2derivatives derivatives application at 25 μM during Chlamydiatrachomatis infection of HeLa229 cells.

EXAMPLES

I. Synthesis of Compounds of Formula (I)

A general scheme for the synthesis of compounds described herein is asfollows (more details can be found in N-methyl dihydroquinazolinonesderivatives of Retro-2 with enhanced efficacy against Shiga toxin., R.Noel, N. Gupta, V. Pons, A. Goudet, M. D. Garcia-Castillo, A. Michau, J.Martinez, D. A. Buisson, L. Johannes, D. Gillet, J. Barbier, J.-C.Cintrat, J. Med. Chem., 2013, 56(8), 3404-3413)

2-(5-(2-(4-fluorophenyl)thiazol-4-yl)thiophen-2-yl)-1-methyl-3-phenyl-2,3-dihydroquinazolin-4(1H)-one(VP89)

To a solution of5-(2-(4-fluorophenyl)thiazol-4-yl)thiophene-2-carbaldehyde (227 mg,0.787 mmol) in THF (5 mL), was added 2-(methylamino)-N-phenylbenzamide(311 mg, 1.377 mmol) and p-toluenesulfonic acid (27 mg, 0.157 mmol). Themixture was refluxed overnight, then neutralized with a saturatedsolution of NaHCO₃ (10 mL) and extracted with ethyl acetate (3×20 mL).The organic layer was concentrated under vacuum, dried over Na₂SO₄,filtrated and concentrated under vacuum. The crude mixture was purifiedby flash chromatography (cyclohexane-ethyl acetate, 1-0 to 7-3)affording the desired compound (165 mg, 42%).

¹H-NMR (400 MHz, DMSO) δ (ppm)=2.99 (s, 3H), 6.51 (s, 1H), 6.78 (d,J=8.1 Hz, 1H), 6.92 (t, J=7.3 Hz, 1H), 6.99 (d, J=3.8 Hz, 1H), 7.26-7.36(m, 5H), 7.38-7.44 (m, 3H), 7.46 (td, J=7.8 Hz, J′=1.6 Hz, 1H), 7.90(dd, J=7.8 Hz, J′=1.6 Hz, 1H), 7.92-7.97 (m, 3H).

¹³C-NMR (100 MHz, DMSO) δ (ppm)=35.5, 76.2, 113.7, 114.4, 116.6, 116.9,117.2, 119.1, 124.2, 126.9, 127.2, 128.6, 128.7, 128.9, 129.0, 129.3,129.5, 129.5, 134.8, 137.8, 140.1, 140.5, 146.8, 149.6, 161.2, 162.5,165.0, 166.5

MS (ESI) [M+H]⁺=498.12

LC/MS (X-bridge 100×4.6 mm)

-   -   Gradient A: t_(R)=24.37 min    -   Gradient D: t_(R)=21.35 min

I.R. (neat, cm⁻¹) 1639, 1601, 1491, 1227, 1156, 742, 691.

II. Biological Activity against Chlamydiales Infections

II.1. Materials and Methods

Cell Lines and Bacteria

HeLa229 (ATCC CCL-2.1) were grown in RPMI1640 medium (Glutamax, 10% FBS,w/o HEPES) (Invitrogen). Stable HeLa229 cell lines were established toconstantly label the Golgi apparatus (B4GalT1 in a pCMV6-AC-mRFP cloningvector, OriGene) and the ER (KDEL in a pDsRed2-ER expression vector).

Simkania negevensis (Sn) strain Z (ATCC VR-1471) was prepared asdescribed previously (Mehlitz A, Karunakaran K, Herweg J A, Krohne G,van de Linde S, Rieck E, Sauer M, Rudel T. The chlamydial organism Snforms ER vacuole contact sites and inhibits ER-stress. Cell Microbiol2014; 16(8):1224-1243).

Briefly, HeLa229 cells were grown to 50-70% confluence, were inoculatedwith Sn in RPMI1640 with 5% FBS, for 6 h at 35° C. in a humidifiedincubator at 5% CO₂. Medium was replaced by infection medium (RPMI1640,Glutamax, 5% FBS, w/o HEPES) and growth was allowed for 3 days. Cellswere mechanically detached and bacteria were released using ˜2-5 mmglass beads (Carl Roth). Low speed supernatant (600×g, 4° C. and 5 min)was subjected to high-speed centrifugation (20,000×g, 4° C. and 30 min)to pellet bacteria. Bacteria were washed twice with 5 ml SPG (250 mMsucrose, 50 mM sodium phosphate, 5 mM glutamate, pH 7.4), aliquoted andstored at −80° C. in SPG.

Chlamydia trachomatis (Ctr). Laboratory-adapted strain L2/434/Bu (ATCCVR902B) was used in assays. Full biological and genetic information isavailable for this strain including complete genome sequence and definedproteome. This strain has a relatively low particle to infectivityratio, perform efficient cell infection and has a higher viability thanstandard genital tract isolates with faster developmental cycle. Cultureconditions have been described in (Wang Y., Kahane S., Cutcliffe L. T.,Skilton R. J., Lambden P. R., Clarke I. N. Development of atransformation system for Chlamydia trachomatis: Restoration of glycogenbiosynthesis by acquisition of a plasmid shuttle vector. PLoS Pathogen,2011, 7(9):e1002258. doi: 10.1371/journal.ppat.1002258).

Sn Infectivity Assays in Presence of Compound According to the PresentInvention

40,000 HeLa cells were seeded in 12-well cluster plates,inhibitor-treated and infected as indicated in the respectiveexperiments.

For infectivity assays cells were either fixed and stained at indicatedtime points (FIG. 2, 4; inclusion formation/primary infection) orbacteria were released via one freeze thaw cycle (−70° C./37° C.)followed by mechanical release through pipetting and transfer to freshHeLa229 cells (1:25-1:50, progeny/infectivity). Cells were centrifugedfor 1 h at 35° C. and medium exchanged to infection medium. Progeny wasfixed at day 3 post infection and processed for staining (FIG. 3) orharvested for immunoblotting (FIG. 1). Infectivity assays were imaged onan automated fluorescence microscope Leica DMIR (FIG. 4). Numbers andaverage sizes of the SCV as well as host cell numbers were determinedvia GroEL and DAPI staining and images were analysed and quantifiedusing FIJI (ImageJ) and Excel (Microsoft).

In progeny assay, bacteria are first grown in HeLa229 cells treated withinhibitors (Retro-2 at a concentration of 25 and 75 μM) and theinfectious particles from this primary infection are applied to freshcells in the absence of inhibitor to measure the bacterial load (GroELimmunoblot) and inclusion formation (immunofluorescence microscopy).

Chlamydia trachomatis (Ctr) Infectivity Assays in Presence of CompoundAccording to the Present Invention

Compound application during Ctr infection. HeLa229 cells were pretreatedwith Retro-2 in concentrations of 25, 50 and 75 μM for 30 min until Ctr(MOI1) were added to the cells. Compounds were present during infection.

(A) Cells with primary infection were lysed 24 h post infection (hpi).To obtain progeny infection compound treated cells were lysed 48 hpi andlysate was used to infect fresh HeLa229 cells. Progeny infection waslysed 24 hpi and analyzed together with primary infection samples byimmunoblot. Chlamydial growth was detected with antibodies againstchlamydial HSP60 protein and Actin was used as loading control (FIG. 6).

(B) Infected cells were fixed with 4% Paraformaldehyde 24 hpi. Cellswere stained by Alexa-680 coupled Phalloidin and Ctr were detected byGFP-signal (FIG. 7).

II.2. Results

Infection by Sn

Tested inhibitor of retrograde transport Retro-2 has had an inhibitireffect on primary and progeny infection for Sn (FIGS. 1, 2, 3 and 4).This was shown by western detection of Sn GroEL (FIG. 1), relative Sninclusion sizes in primary infection (FIG. 2), relative Sn inclusionnumber in progeny infection (FIG. 3), fluorescence microscopy (FIG. 4)and transmission electron microscopy (TEM; FIG. 5).

The maximal inhibition of Sn replication was observed at a concentrationof 75 μM (FIGS. 1, 2, 3 and 4).

Sn inclusions formed normally in DMSO-treated control cells. Retro-2affected the Sn inclusions morphology since sub-vacuolar membranes werehighly enlarged and contained excessive membrane material, probably as aconsequence of defective membrane fusion and vacuole formation (FIG. 5).Parts of the enlarged sub-vacuoles contained just few bacteria locatedat the inclusion boundary which may indicate defective Sn replication.Additionally, sub-vacuoles were surrounded by multiple membranes atseveral areas indicative of a role of Retro-2 inhibited pathways in Sninclusions formation and membrane fusion defect.

In summary, Retro-2 compound inhibits primary and progeny infection forSn.

Infection by Chlamydia trachomatis

Experiments performed to test the anti-chlamydial activity of Retro-2demonstrated a strong inhibitory effect of Retro-2 on Ctr development(FIGS. 6 and 7). This was shown by western detection of Ctr Hsp60 ininfected cells (FIG. 6) and staining of Ctr inclusions withGFP-expressing Ctr (FIG. 7).

This treatment of the primary Ctr infection also resulted in significantconsequences of the progeny infection (FIG. 6); Retro-2 which blockschlamydial replication in the primary infection consequently showshighly reduced amounts of Ctr in the progeny with almost no Ctr Hsp60detection at 75 μM of Retro-2.

These results highlight the utility of Retro-2 as an anti-chlamydialcompound. The results depicted in FIGS. 6 and 7 reveal that Retro-2affects different cellular mechanisms and have various targets which areimportant at defined stages of the chlamydial developmental cycle.

III. Biological Activity against Chlamydiales Infections

III.1. Materials and Methods

Cell Lines and Bacteria

HeLa229 (ATCC CCL-2.1). Cells were grown in RPMI1640 medium (Glutamax,10% FBS, w/o HEPES) (Invitrogen).

Chlamydia trachomatis (Ctr). Laboratory-adapted strain L2/434/Bu (ATCCVR902B) was used in assays. Full biological and genetic information isavailable for this strain including complete genome sequence and definedproteome. This strain has a relatively low particle to infectivityratio, perform efficient cell infection and has a higher viability thanstandard genital tract isolates with faster developmental cycle. Cultureconditions have been described in (Wang Y., Kahane S., Cutcliffe L. T.,Skilton R. J., Lambden P. R., Clarke I. N. Development of atransformation system for Chlamydia trachomatis: Restoration of glycogenbiosynthesis by acquisition of a plasmid shuttle vector. PLoS Pathogen,2011, 7(9):e1002258. doi: 10.1371/journal.ppat.1002258).

Chlamydia trachomatis (Ctr) Infectivity Assays in Presence of CompoundsAccording to the Present Invention

Compound application during Chlamydia trachomatis infection. HeLa229cells were pretreated with Retro-2 and Retro-2 derivatives inconcentrations of 25 μM for 1 hour until Chlamydia trachomatis (MOI1)were added to the cells. Compounds were present during infection. Toobtain progeny infection, compound treated cells were lysed 24 h postinfection (hpi) and lysate was used to infect fresh HeLa 229 cells.Infected cells of progeny infection were fixed with 4% Paraformaldehyde48 hpi. Cells were stained for DAPI and Chlamydia trachomatis weredetected by GFP-signal. Images are representative of n=2 independentexperiments. Quantification of infected cells by Chlamydia trachomatiswas realized from microscopy images with Image J software (FIG. 8) andcellular protection by compounds at 25 μM was then determined (Table 1)by comparison with solvent-treated cells (control) with the followingequation:

${{Cellular}\mspace{14mu}{protection}} = {100 - {\frac{\%\mspace{14mu}{of}\mspace{14mu}{infected}\mspace{14mu}{cells}\mspace{14mu}{in}\mspace{14mu}{presence}\mspace{14mu}{of}\mspace{14mu}{inhibitor}}{\%\mspace{14mu}{of}\mspace{14mu}{infected}\mspace{14mu}{cells}\mspace{14mu}{in}\mspace{14mu}{control}} \times 100}}$III.2. Results

TABLE 1 Cellular protection at 25 μM (%) Retro-2 18.1 Retro-2.1 100RN-2-016 91.8 RN-3-072 100 Compound 25 100 VP-89 88.5

This treatment of the primary Ctr infection with Retro-2 derivativesresulted in a strong consequences of the progeny infection at 25 μM incomparison with Retro-2 (FIG. 8).

Conclusion

These results highlight the utility of Retro-2 derivatives of formula(I′) as an anti-chlamydial compound. The results depicted in FIG. 8 thatRetro-2 derivatives are much more potent than Retro-2 at lowconcentration and affect cellular mechanisms that are important atdefined stages of the chlamydial developmental cycle.

The invention claimed is:
 1. A method for treating a Chlamydiaeinfection comprising the administration of a therapeutically effectiveamount of a compound of formula (I) to a subject in need thereof

Wherein: R₁ is at each occurrence, independently selected from H, Cl,Br, I, F; R₂ is independently selected from Br and 5 to 10 memberedheteroaryl, said 5 to 10 membered heteroaryl groups being optionallysubstituted by one to three R₅; R₃ is independently selected from H, F,Cl, Br, I, C₁-C₆ alkyl; R₄ is, at each occurrence, independentlyselected from H, C₁-C₆ alkyl; R₅ is independently selected from F, Cl,Br, I, C₁-C₆ alkyl, C₆-C₁₀ aryl, said aryl group being optionallysubstituted by one to three R₆; R₆ is F, Cl, Br, I; p is 1, 2 or 3; andthe stereoisomeric forms, mixtures of stereoisomeric forms orpharmaceutically acceptable salts forms thereof, providing said compoundis not of the following formula:


2. The method of claim 1, wherein the Chlamydiae infection is aChlamydia or Simkania infection.
 3. The method of claim 1, wherein atleast one of R₁ is H or F.
 4. The method of claim 1, wherein R₂ is a 5to 10 membered heteroaryl.
 5. The method of claim 4, wherein R₂ is1,3-thiazolyl.
 6. The method of claim 1, which is selected from thegroup consisting of:6-fluoro-1-methyl-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one;2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one;2-(5-bromothiophen-2-yl)-1-methyl-3-phenyl-2,3-dihydroquinazolin-4(1H)-one;3-(2-chlorophenyl)-1-methyl-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-2,3-dihydroquinazolin-4(1H)-one;and2-(5-(2-(4-fluorophenyl)thiazol-4-yl)thiophen-2-yl)-1-methyl-3-phenyl-2,3-dihydroquinazolin-4(1H)-one.7. A pharmaceutical composition comprising a compound of formula (Ia):

Wherein: R₁, R₃, R₄, R₆, and p are as defined in formula (I) in claim 1,R₄ is independently selected from H or C₁-C₆ alkyl, and thestereoisomeric forms, mixtures of stereoisomeric forms orpharmaceutically acceptable salts forms thereof, in admixture with oneor more pharmaceutically acceptable excipients.
 8. The pharmaceuticalcomposition of claim 7, wherein R₄ is C₁-C₆ alkyl, notably methyl. 9.The pharmaceutical composition of claim 7, wherein R₃ is H.
 10. Thepharmaceutical composition of claim 7, wherein R₁ is H.
 11. Thepharmaceutical composition of claim 7, wherein the compound of formula(Ia) is2-(5-(2-(4-fluorophenyl)thiazol-4-yl)thiophen-2-yl)-1-methyl-3-phenyl-2,3-dihydroquinazolin-4(1H)-one(VP89).
 12. A compound of formula (Ia):

Wherein: R₁, R₃, R₄, R₆, and p are as defined in formula (I) in claim 1,R₄ is independently selected from H or C₁-C₆ alkyl, and thestereoisomeric forms, mixtures of stereoisomeric forms orpharmaceutically acceptable salts forms thereof.
 13. The method of claim4, wherein R₂ is 4-(1,3-thiazolyl).